7. Project Summary/Abstract The proposal is focused on understanding the functions of the extremely conserved multi-protein exon junction complex (EJC) in specifying parallel nonsense-mediated mRNA decay (NMD) pathways. NMD is as an essential post-transcriptional mechanism that regulates normal gene expression and also serves a quality control function. A detailed understanding of these processes is crucial for betterment of human health as mutations that disrupt the EJC and NMD proteins cause developmental defects, intellectual disability and mental retardation. The EJC is deposited upstream of mRNA exon-exon junctions by the spliceosome, and has major consequences on downstream mRNA metabolism. An EJC downstream of a termination codon is widely accepted as an absolute mark for premature translation termination and an NMD trigger. The current proposal is motivated by two unexpected discoveries that challenge the dogmatic view of EJC composition and function. First, we recently revealed in vivo EJC occupied sites by developing a novel high-throughput approach termed RIPiT-Seq that purifies RNPs containing a distinct pair of proteins and identifies their transcriptome-wide footprints. This demonstrated that the EJC is not detected at all predicted binding sites and, frequently, the complex is also detected at unexpected positions. We now show that the EJC composition also varies from position to position, and there exist in human cells at least two mutually exclusive or 'alternate' EJCs, that contain different protein factors. Notably, the two alternate EJC factors, RNPS1 and MLN51 were described previously as unique components of parallel NMD branches. We show that the two alternate EJC factors have distinct NMD targets, and they interact differentially with proteins of the parallel NMD branches. Our overall hypothesis is that the alternate EJCs control distinctly different biological activities in the cell. We propose to use a multi-disciplinary approach to discover how different EJC compositions are connected to parallel NMD branches and what biological activities are regulated by each unique complex. In Aim 1, we will use RNA-Seq and RIPiT-Seq approaches to define the alternate EJC-regulated NMD targets, their distinctive features, and hence the unique biological functions of alternate EJCs. In Aim 2, we will use a metabolic labeling approach to address a largely neglected question regarding the kinetics and order of events during EJC remodeling. Using well-established strategies to enhance and/or inhibit NMD at different steps, we will investigate when and where in the NMD pathway do alternate EJCs function. In Aim 3, we will use reporters from parallel NMD branches to test the relationship between alternate EJCs and parallel NMD pathways. We will also use RNA- Seq and RIPiT-Seq based global analyses to reveal specific mRNA and protein interactions of alternate EJC- Upf complexes in parallel NMD branches. Our goal is to reveal the target specificity, underlying mechanisms and cellular functions of parallel NMD branches.